title: Small GTPase Rab14 down-regulates UT-A1 urea transport activity through enhanced clathrin-dependent endocytosis.
abstract: The UT-A1 urea transporter plays an important role in the urinary concentration mechanism. However, the molecular mechanisms regarding UT-A1 trafficking, endocytosis, and degradation are still unclear. In this study, we identified the small GTPase Rab14 as a binding partner to the C terminus of UT-A1 in a yeast 2-hybrid assay. Interestingly, UT-A1 binding is preferential for the GDP-bound inactive form of Rab14. Coinjection of Rab14 in Xenopus oocytes results in a decrease of UT-A1 urea transport activity, suggesting that Rab14 acts as a negative regulator of UT-A1. We subsequently found that Rab14 reduces the cell membrane expression of UT-A1, as evidenced by cell surface biotinylation. This effect is blocked by chlorpromazine, an inhibitor of the clathrin-mediated endocytic pathway, but not by filipin, an inhibitor of the caveolin-mediated endocytic pathway. In kidney, Rab14 is mainly expressed in IMCD epithelial cells with a pattern identical to UT-A1 expression. Consistent with its role in participating in clathrin-mediated endocytosis, Rab14 localizes in nonlipid raft microdomains and codistributes with Rab5, a marker of the clathrin-mediated endocytic pathway. Taken together, our study suggests that Rab14, as a novel UT-A1 partner, may have an important regulatory function for UT-A1 urea transport activity in the kidney inner medulla.
authors.affiliation: 1Department of Physiology, Emory University School of Medicine, 615 Michael St., Atlanta, GA 30322, USA. gchen3@emory.edu.
authors.forename: Hua
authors.initials: H
authors.lastname: Su
authors.forename: Bingchen
authors.initials: B
authors.lastname: Liu
authors.forename: Otto
authors.initials: O
authors.lastname: Fröhlich
authors.forename: Heping
authors.initials: H
authors.lastname: Ma
authors.forename: Jeff M
authors.initials: JM
authors.lastname: Sands
authors.forename: Guangping
authors.initials: G
authors.lastname: Chen
mesh_terms: D000818:Animals; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes; D034622:RNA Interference; D034741:RNA, Small Interfering; D051381:Rats; D020798:Two-Hybrid System Techniques; D014508:Urea / Q000378:metabolism*; D014981:Xenopus; D020691:rab GTP-Binding Proteins; D000096982:Urea Transporters
chemical_list: D002746:Chlorpromazine; D002966:Clathrin; D026901:Membrane Transport Proteins; D034741:RNA, Small Interfering; D014508:Urea; D020691:rab GTP-Binding Proteins; D000096982:Urea Transporters; C553411:Rab14 protein, human
keywords: inner medulla; membrane protein; protein interaction